In general, the growth and/or culture of animal cells, especially mammalian cells requires a constant supply of oxygen and effective removal of gaseous metabolic by-products, mainly carbon dioxide. The requisite gas exchange can be accomplished in a number of ways, including agitation of the reaction vessel and bubbling of oxygen-containing gases through the culture. In order for agitation to be effective, very low volumes of culture medium must be used since the effectiveness of this method depends on exposure of all of the cells in the culture to the surface. Bubbling of gases obviates this problem to some extent, but superimposes another wherein sufficient bubbling to be effective creates shear forces believed to be harmful to the relatively delicate animal cell membranes. Culture vessels, which specifically do not require agitation and provide the supply of oxygen through the use of gas bubbles, are, for instance, described in U.S. Pat. No. 4,259,449.
A different approach to the supply of needed oxygen and removal of gases is described in Miltenburger et al., U.S. Pat. No. 4,649,114, which describes the supply of oxygen and removal of carbon dioxide in a fermentation vessel for animal cells, especially insect cells, using a tube or hose of a synthetic polymer such as silicone rubber, laminated silicone rubber products, or a polytetrafluoroethylene (Teflon.RTM.) to provide the oxygen through diffusion. The tubes described by Miltenburger are required to be nonadherent with regard to the cells, thick enough to provide mechanical strength, and thin enough to permit oxygen to pass through readily. It is required that the oxygen be supplied without bubbling. The oxygen-dispensing tube is wound around a support in the illustrated embodiments and is stated to be successful in providing sufficient oxygen in the illustrated reaction vessels, which require agitation of the culture medium through mechanical or other means.
In variations of the method to supply oxygen through a membrane, the hollow fiber approach described in U.S. Pat. No. 4,391,912 evidently relies on the supply of oxygen along with culture medium which is provided through a closely spaced series of hollow fibers interspersed among the suspended cell culture. Similarly, U.S. Pat. No. 3,997,396 describes the supply of oxygen through a fiber to cells attached to its outer surface. In both of these cases, the proximity of the cells to be supplied oxygen to the membrane is relied upon to assure adequate transfer.
The continuous supply of medium and withdrawal of secreted products from animal cells cultured in an arrested state of proliferation and provision of oxygen through a tubular semipermeable membrane disposed throughout the culture medium is described in U.S. Pat. No. 4,537,860. The cell culture method described utilizes silicone rubber tubing, typically 1 mm inside diameter and 2 mm outside diameter, to supply oxygen to the culture. Again, sufficient surface area relative to the volume of the culture chamber must be assured so that all cells are proximal to the membrane.
Despite the successful operation of some of the culture methods described in the art, difficulty with providing an adequate supply of oxygen to desirably large volumes of cell culture remains. In general, it is found that the transfer of oxygen-containing gas from the interior of the supply tubular membrane to the culture medium is not as efficient as desired. This may be due in part to a limited surface area, but may also be due to other physical constraints such as an accumulated filmlike barrier residing at the surface of the membrane. It is not clear which factors account for limitations on the capacity of the membrane to transfer oxygen to the cellular environment.